Cancer predispositions in humans and severe phenotypes in yeast result from defects in the Mre11/ Rad50/Nbs1 (MRN) complex. The Mre11 nuclease complex with the Rad50 ATPase is conserved from archaea to humans and regulated by Nbs1 in S. pombe and higher eukaryotes. MRN plays central and essential roles in repairing DMA double-strand breaks (DSBs) during homologous recombination repair (HRR) as well as acting in meiosis, antibody hypermutation, telomere maintenance, and DMA damage signaling through ATM kinase. Detailed mechanistic insights into these diverse MRN functions are limited. We therefore propose two specific aims to further knowledge of MRN structural biochemistry, interactions, and conformational changes relevant to MRN DNA damage repair and signaling functions. To accomplish these aims, we will apply advanced biophysical techniques, including synchrotron solution x-ray scattering and atomic resolution crystal structure technologies in concert with genetic and mutational analyses in yeast. The expected results will characterize functionally key Mre11 and Rad50 protein:protein and protein:DNA interfaces, conformations, and interaction architectures. The proposed coupled biophysical and genetic studies will test hypotheses regarding Mre11's role in DNA target specificity and processing, RadSO's role in ATP-induced conformational controls and architectural interactions, and the role of the Nbs1 C-terminal domain in modulating Mre11 and RadSO activities. Furthermore, the DNA damage sensitivity observed in the absence of any MRN complex member makes these structures a basis for the development of inhibitors to increase cellular sensitivity to ionizing radiation and other DNA damaging agents as adjuvants in cancer radiotherapy and chemotherapy. Together, the proposed experiments will therefore provide results important for a unified understanding of the molecular mechanisms underlying the roles of MNR complexes in genetic integrity and cancer resistance as well as MRN structures as potential targets for cancer therapeutics.